Process for correction of a disulfide misfold in Fc molecules

ABSTRACT

The present invention concerns a process by which a misfold in an Fc fusion molecule can be prevented or corrected. In one embodiment, the process comprises (a) preparing a pharmacologically active compound comprising an Fc domain; (b) treating the fusion molecule with a copper (II) halide; and (c) isolating the treated fusion molecule. The pharmacologically active compound can be an antibody or a fusion molecule comprising a pharmacologically active domain and an Fc domain. The preferred copper (II) halide is CuCl 2 . The preferred concentration thereof is at least about 10 mM for fusion molecules prepared in  E. coli ; at least about 30 mM for fusion molecules prepared in CHO cells. The process can be employed with any number of pharmacologically active domains. Preferred pharmacologically active domains include OPG proteins, leptin proteins, soluble portions of TNF receptors (e.g., wherein the fusion molecule is etanercept), IL-1ra proteins, and TPO-mimetic peptides. The Fc domain preferably has a human sequence, with an Fc sequence derived from IgG1 most preferred. An exemplary Fc sequence is shown in FIG.  5  hereinafter.

The present application is a divisional application of U.S. patentapplication Ser. No. 09/709,704 (now U.S. Pat. No. 6,808,902), which wasfiled Nov. 9, 2000 and claimed benefit of priority of U.S. ProvisionalApplication No. 60/165,188, filed Nov. 12, 1999

BACKGROUND OF THE INVENTION

Recombinant proteins are an emerging class of therapeutic agents. Suchrecombinant therapeutics have engendered advances in protein formulationand chemical modification. Such modifications can protect therapeuticproteins, primarily by blocking their exposure to proteolytic enzymes.Protein modifications may also increase the therapeutic protein'sstability, circulation time, and biological activity. A review articledescribing protein modification and fusion proteins is Francis (1992),Focus on Growth Factors 3:4-10 (Mediscript, London), which is herebyincorporated by reference.

One useful modification is combination with the “Fc” domain of anantibody. Antibodies comprise two functionally independent parts, avariable domain known as “Fab”, which binds antigen, and another domainknown as “Fc”, which links to such effector functions as complementactivation and attack by phagocytic cells. An Fc has a long serumhalf-life, whereas an Fab is short-lived. Capon et al. (1989), Nature337:525-31. When constructed together with a therapeutic protein, an Fcdomain can provide longer half-life or incorporate such functions as Fcreceptor binding, protein A binding, complement fixation and perhapseven placental transfer. Id. Table 1 summarizes use of Fc fusions knownin the art.

TABLE 1 Fc fusion with therapeutic proteins Fusion Therapeutic Form ofFc partner implications Reference IgG1 N-terminus of Hodgkin's disease;U.S. Pat. No. CD30-L anaplastic lymphoma; T- 5,480,981 cell leukemiaMurine Fcγ2a IL-10 anti-inflammatory; Zheng et al. (1995), J. transplantrejection Immunol. 154: 5590-600 IgG1 TNF receptor septic shock Fisheret al. (1996), N. Engl. J. Med. 334: 1697- 1702; Van Zee, K. et al.(1996), J. Immunol. 156: 2221-30 IgG, IgA, TNF receptor inflammation,autoimmune U.S. Pat. No. 5,808,029, IgM, or IgE disorders issued Sep.15, (excluding 1998 the first domain) IgG1 CD4 receptor AIDS Capon etal.(1989), Nature 337: 525-31 IgG1, N-terminus anti-cancer, antiviralHarvill et al. (1995), IgG3 of IL-2 Immunotech. 1: 95-105 IgG1C-terminus of osteoarthritis; WO 97/23614, published OPG bone densityJul. 3, 1997 IgG1 N-terminus of anti-obesity PCT/US 97/23183, filedleptin Dec. 11, 1997 Human Ig CTLA-4 autoimmune disorders Linsley(1991), J. Exp. Cγ1 Med. 174: 561-9

Despite their advantages, use of Fc fusion molecules may be limited bymisfolding upon expression in a desired cell line. Such misfolded Fcfusion molecules may generate an immune response in vivo or may causeaggregation or stability problems in production.

SUMMARY OF THE INVENTION

The present invention concerns a process by which a misfold in an Fcfusion molecule can be prevented or corrected. In one embodiment, theprocess comprises:

-   -   (a) preparing a fusion molecule comprising (i) a        pharmacologically active domain and (ii) an Fc domain;    -   (b) treating the fusion molecule with a copper (II) halide; and    -   (c) isolating the treated fusion molecule.

The preferred copper (II) halide is CuCl₂. The preferred concentrationthereof is at least about 10 mM for fusion molecules prepared in E.coli; at least about 30 mM for fusion molecules prepared in CHO cells.

An alternative embodiment of the process comprises the following steps:

-   -   (a) preparing a fusion molecule comprising (i) a        pharmacologically active domain and (ii) an Fc domain;    -   (b) treating the fusion molecule with guanidine HCl at a        concentration of at least about 4M;    -   (c) increasing the pH to about 8.5; and    -   (d) isolating the treated fusion molecule.

Each of these processes can be employed with any number ofpharmacologically active domains. Preferred pharmacologically activedomains include OPG proteins, leptin proteins, TNF-α inhibitors (e.g.,wherein the fusion molecule is etanercept), IL-1 inhibitors (e.g.,IL-1ra proteins, which are preferred), and TPO-mimetic peptides. Alsowithin the claimed process are molecules in which the pharmacologicallyactive compound is an antibody. The Fc domain preferably has a humansequence, with an Fc sequence derived from IgG1 most preferred. Anexemplary Fc sequence is shown in FIG. 5 hereinafter.

Although mostly contemplated as therapeutic agents, compounds of thisinvention may also be useful in screening for such agents. For example,one could use an Fc-peptide (e.g., Fc-SH2 domain peptide) in an assayemploying anti-Fc coated plates. The vehicle, especially Fc, may makeinsoluble peptides soluble and thus useful in a number of assays.

The compounds prepared by the process of this invention may be used fortherapeutic or prophylactic purposes by formulating them withappropriate pharmaceutical carrier materials and administering aneffective amount to a patient, such as a human (or other mammal) in needthereof.

Numerous additional aspects and advantages of the present invention willbecome apparent upon consideration of the figures and detaileddescription of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Reversed-phase high performance liquid chromatography (RP-HPLC)chromatogram showing Fc-OPG prepared in E. coli with and withouttreatment with 10 mM CuCl₂. At a concentration of 10 mM, CuCl₂ causescomplete irreversible elimination of the RP post-peak.

FIG. 2. RP-HPLC chromatogram showing Fc-OPG prepared in E. coli treatedwith CDAP. The RP post-peak molecular weight increased by 52 Da.

FIG. 3. RP-HPLC chromatogram showing Fc-OPG prepared in E. coli treatedwith CDAP and subsequently subjected to base cleavage. The base cleavagereveals that cysteines 148 and 206 were labeled by CDAP.

FIG. 4. RP-HPLC chromatogram showing Fc-OPG prepared in CHO cells, withand without treatment with 30 mM CuCl₂. At a concentration of 30 mM,CuCl₂ causes complete irreversible elimination of the RP post-peak.

FIG. 5. Exemplary nucleic acid and amino acid sequences (SEQ ID NOS: 1and 2, respectively) of a human IgG1 Fc that may be used in thisinvention.

FIG. 6. Reaction scheme showing cyanylation of cysteine residues inacidic conditions with CDAP.

FIG. 7. Reaction scheme showing cleavage of the cyanylated cysteine byammonia. Under alkaline and denaturing conditions (1.5N NH₄OH and 4MGdHCl), the protein backbone undergoes cleavage at the carbonyl-amidepeptide bond on the cyanylated cysteine. The result is an amidatedN-terminal peptide and an iminothiazolidine (ITZ) peptide which areseparated and identified by LC-MS.

FIG. 8. RP-HPLC chromatograms with mass spectrometry determinedmolecular weights of Fc-OPG (A), Fc-OPG reacted with CuCl₂ (B), andFc-OPG reacted with CDAP followed by CuCl₂ (C).

FIG. 9. RP-HPLC chromatograms of purified main and isoform peaksfollowing CDAP reaction and cleavage.

FIG. 10. Schematic diagram of the Fc-OPG construct, CDAP-cleavagepattern and the missing Fc-domain disulfide linkage that results in theRP-HPLC isoform.

FIG. 11. Reversed-phase HPLC of untreated (upper trace) andcopper-treated (lower trace) Fc-OPG.

FIG. 12. Size-exclusion HPLC of Fc-OPG after 2 years incubation at 29°C.; copper-treated Fc-OPG (lower trace) and untreated Fc-OPG (uppertrace).

DETAILED DESCRIPTION OF THE INVENTION In General

In production of therapeutic proteins by recombinant techniques, thetherapeutic protein quite often must be refolded to an activeconformation. At present, the refold process includes an oxidation stepto form the disulfide structure of the produced recombinant protein. Thereagents commonly used to catalyze formation of the disulfide structureare the free amino acids cysteine/cystamine at a pH of 8-9. In addition,copper sulfate at a copper concentration in the micromolar range canalso be used. For refolding of Fc fusion molecules, however, coppersulfate cannot be used due to the high levels of arginine (0.5M) whichis a chelator of Cu⁺⁺. Remaining misfolds are normally removed duringthe subsequent purification process. After process purification ofFc-OPG, for example, a post-peak was isolated by reversed-phase (RP)high performance liquid chromatography (HPLC). Data (primarily frompeptide maps and Ellmann's reagent) suggested the RP post-peak did notcontain free thiols.

In the course of formulation development, the effects of mono- anddivalent cations can be related to increases in various chemicaldegradations. During the screening of Fc-OPG, it was observed that 1 mMCuCl₂ had a dramatic effect at reducing the amount of the RP post-peak.Subsequent experiments have shown that 10 mM CuCl₂ is required for thecomplete irreversible elimination of the RP post-peak (see FIG. 1). TheRP post-peak was shown to result from an unpaired disulfide through theuse of the reagent 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate(CDAP). For each free sulfhydryl, CDAP adds a CN group or increases themolecular weight by 26 Da. Treating Fc-OPG with CDAP and subsequentHPLC/MS analysis indicated that the molecular weight of the Fc-OPG RPpost-peak increased by 52 Da or the equivalent of two sulfhydryl groupslabeled (see FIG. 2). The molecular weight of the RP main-peak did notchange after treating with CDAP. Subsequent base cleavage of thecyanylated RP post-peak indicated that only cysteines 148 and 206 (FIG.3) are labeled by CDAP. The disulfide, cys148-cys206, which is notformed in the RP post-peak is found in the CH3 region which is thesecond disulfide loop of Fc and is the same disulfide that was difficultto form in Fc-Leptin. Further, pretreatment with CuCl₂ followed bylabeling with CDAP produces no detectable change in molecular weight,supporting the conclusion that Cu⁺⁺ fixes the disulfide problem inFc-OPG. Similar results utilizing CuCl₂ have been observed for theCHO—OPG-Fc (FIG. 4) and allows us to conclude that all of ourrecombinant Fc molecules have the same difficulty in forming thedisulfide in the CH3 region of Fc. Surprisingly, CHO cells have the samedifficulty in Fc disulfide formation to approximately the same degree.

Alternatively the process could be effected as a post-refold treatment.Several methods can be used to eliminate the RP post-peak. In additionto the CuCl₂ treatment, a similar removal of post-peak is observed afterdenaturing in a minimum of 4 M guanidine HCl and increasing the pH from5.0 to 8.5. Both treatments strongly support that free thiols areinvolved which can be converted to a disulfide.

DEFINITION OF TERMS

The terms used throughout this specification are defined as follows,unless otherwise limited in specific instances.

The term “comprising” means that a compound may include additional aminoacids on either or both of the N- or C-termini of the given sequence. Ofcourse, these additional amino acids should not significantly interferewith the activity of the compound.

The term “native Fc” refers to molecule or sequence comprising thesequence of a non-antigen-binding fragment resulting from digestion ofwhole antibody, whether in monomeric or multimeric form. The originalimmunoglobulin source of the native Fc is preferably of human origin andmay be any of the immunoglobulins, although IgG1 and IgG2 are preferred.Native Fc's are made up of monomeric polypeptides that may be linkedinto dimeric or multimeric forms by covalent (i.e., disulfide bonds) andnon-covalent association. The number of intermolecular disulfide bondsbetween monomeric subunits of native Fc molecules ranges from 1 to 4depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2,IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bondeddimer resulting from papain digestion of an IgG (see Ellison et al.(1982), Nucleic Acids Res. 10:4071-9). The term “native Fc” as usedherein is generic to the monomeric, dimeric, and multimeric forms.

The term “Fc variant” refers to a molecule or sequence that is modifiedfrom a native Fc but still comprises a binding site for the salvagereceptor, FcRn. International applications WO 97/34631 (published 25Sep. 1997) and WO 96/32478 describe exemplary Fc variants, as well asinteraction with the salvage receptor, and are hereby incorporated byreference. Thus, the term “Fc variant” comprises a molecule or sequencethat is humanized from a non-human native Fc. Furthermore, a native Fccomprises sites that may be removed because they provide structuralfeatures or biological activity that are not required for the fusionmolecules of the present invention. Thus, the term “Fc variant”comprises a molecule or sequence that lacks one or more native Fc sitesor residues that affect or are involved in (1) disulfide bond formation,(2) incompatibility with a selected host cell (3) N-terminalheterogeneity upon expression in a selected host cell, (4)glycosylation, (5) interaction with complement, (6) binding to an Fcreceptor other than a salvage receptor, or (7) antibody-dependentcellular cytotoxicity (ADCC). Fc variants are described in furtherdetail hereinafter.

The term “Fc domain” encompasses native Fc and Fc variant molecules andsequences as defined above. As with Fc variants and native Fc's, theterm “Fc domain” includes molecules in monomeric or multimeric form,whether digested from whole antibody or produced by other means.

The term “multimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two or more polypeptide chainsassociated covalently, noncovalently, or by both covalent andnon-covalent interactions. IgG molecules typically form dimers; IgM,pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, ortetramers. Multimers may be formed by exploiting the sequence andresulting activity of the native Ig source of the Fc or by derivatizing(as defined below) such a native Fc.

The term “dimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two polypeptide chains associatedcovalently or non-covalently.

The terms “derivatizing” and “derivative” or “derivatized” compriseprocesses and resulting compounds respectively in which (1) the compoundhas a cyclic portion; for example, cross-linking between cysteinylresidues within the compound; (2) the compound is cross-linked or has across-linking site; for example, the compound has a cysteinyl residueand thus forms cross-linked dimers in culture or in vivo; (3) one ormore peptidyl linkage is replaced by a non-peptidyl linkage; (4) theN-terminus is replaced by —NRR¹, NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹,—NHC(O)NHR, a succinimide group, or substituted or unsubstitutedbenzyloxycarbonyl-NH—, wherein R and R¹ and the ring substituents are asdefined hereinafter; (5) the C-terminus is replaced by —C(b)R² or —NR³R⁴wherein R², R³ and R⁴ are as defined hereinafter; and (6) compounds inwhich individual amino acid moieties are modified through treatment withagents capable of reacting with selected side chains or terminalresidues. Derivatives are further described hereinafter.

The term “peptide” refers to molecules of 3 to 40 amino acids, withmolecules of 3 to 20 amino acids preferred and those of 6 to 15 aminoacids most preferred. Exemplary peptides may be randomly generated byany of the methods cited above, carried in a phage display library, orderived by digestion of proteins.

The term “native protein” refers to a molecule having an amino acidsequence that may be isolated from an organism without modification byrecombinant DNA techniques or other methods.

The term “protein fragment” refers to a molecule or sequence thatcomprises only a portion of the sequence of a native protein but stillretains the pharmacological activity of interest. The term “proteinfragment” thus comprises, for example, the soluble domain of a cellularreceptor (e.g., a receptor for tumor necrosis factor).

The term “protein variant” refers to a molecule or sequence that ismodified from a native protein but still retains the pharmacologicalactivity of interest. Thus, the term “protein variant” comprises amolecule or sequence in which non-native residues substitute for nativeresidues, non-native residues are added, or native residues are deleted.Any native residue may be removed because it provides structuralfeatures or biological activity that are not required for thepharmacological activity of interest of the fusion molecules of thepresent invention. Thus, the term “protein variant” comprises a moleculeor sequence that lacks one or more native protein sites or residues thataffect or are involved in (1) intracellular signaling, (2)incompatibility with a selected host cell (3) N-terminal heterogeneityupon expression in a selected host cell, (4) glycosylation, (5)interaction with other proteins (e.g., dimerization domains), (6)binding to a receptor or other protein that does not affect thepharmacological activity of interest, or (7) antibody-dependent cellularcytotoxicity (ADCC). Fc variants are described in further detailhereinafter.

The terms “derivatizing” and “derivative” or “derivatized” as used withrespect to proteins refers to proteins in which (1) the protein ismodified to include a cyclic portion; for example, cross-linking betweencysteinyl residues within the compound; (2) the compound is cross-linkedor has a cross-linking site; for example, the compound has a cysteinylresidue and thus forms cross-linked dimers in culture or in vivo; (3)one or more peptidyl linkage is replaced by a non-peptidyl linkage; (4)the N-terminus is replaced by —NRR¹, NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹,—NHC(O)NHR, a succinimide group, or substituted or unsubstitutedbenzyloxycarbonyl-NH—, wherein R and R¹ and the ring substituents are asdefined hereinafter; (5) the C-terminus is replaced by —C(O)R² or —NR³R⁴wherein R², R³ and R⁴ are as defined hereinafter; and (6) compounds inwhich individual amino acid moieties are modified through treatment withagents capable of reacting with selected side chains or terminalresidues. Derivatives are further described hereinafter.

The term “polypeptide” refers to native proteins, protein variants,protein derivatives, and protein fragments.

The term “pharmacologically active” means that a substance so describedis determined to have activity that affects a medical parameter (e.g.,blood pressure, blood cell count, cholesterol level) or disease state(e.g., cancer). Thus, pharmacologically active peptides compriseagonistic or mimetic and antagonistic peptides as defined below.

The term “OPG protein” refers collectively to the novel member of thetumor necrosis factor receptor family described in International patentapplication WO 97/23614. Exemplary OPG proteins are polypeptidescomprising the rat, mouse or human OPG sequences or a consensus of therat, mouse and human sequences.

The term “TNF-α inhibitor” thus includes solubilized TNF receptors,antibodies to TNF, antibodies to TNF receptor, inhibitors of TNF-αconverting enzyme (TACE), and other molecules that affect TNF activity.

TNF-α inhibitors of various kinds are disclosed in the art, includingthe following references:

-   -   European patent applications 308 378; 422 339; 393 438; 398 327;        412 486; 418 014, 417 563, 433 900; 464 533; 512 528; 526 905;        568 928; 663 210; 542 795; 818 439; 664 128; 542 795; 741 707;        874 819; 882 714; 880 970; 648 783; 731 791; 895 988; 550 376;        882 714; 853 083; 550 376; 943 616;

U.S. Pat. Nos. 5,136,021; 5,929,117; 5,948,638; 5,807,862; 5,695,953;5,834,435; 5,817,822; 5,830,742; 5,834,435; 5,851,556; 5,853,977;5,359,037; 5,512,544; 5,695,953; 5,811,261; 5,633,145; 5,863,926;5,866,616; 5,641,673; 5,869,677; 5,869,511; 5,872,146; 5,854,003;5,856,161; 5,877,222; 5,877,200; 5,877,151; 5,886,010; 5,869,660;5,859,207; 5,891,883; 5,877,180; 5,955,480; 5,955,476; 5,955,435.

International (WO) patent applications 90/13575, 91/03553, 92/01002,92/13095, 92/16221, 93/07863, 93/21946, 93/19777, 95/34326, 96/28546,98/27298, 98/30541, 96/38150, 96/38150,97/18207, 97/15561, 97/12902,96/25861, 96/12735, 96/11209, 98/39326, 98/39316, 98/38859, 98/39315,98/42659, 98/39329, 98/43959, 98/45268, 98/47863, 96/33172, 96/20926,97/37974, 97/37973, 96/35711, 98/51665, 98/43946, 95/04045, 98/56377,97/12244, 99/00364, 99/00363, 98/57936, 99/01449, 99/01139, 98/56788,98/56756, 98/53842, 98/52948, 98/52937, 99/02510, 97/43250, 99/06410,99/06042, 99/09022, 99/08688, 99/07679, 99/09965, 99/07704, 99/06041,99/37818, 99/37625, 97/11668;

Japanese (JP) patent applications 10147531, 10231285, 10259140, and10130149, 10316570, 11001481, and 127,800/1991;

German (DE) application 19731521;

British (GB) applications 2 218 101, 2 326 881, 2 246 569.

The disclosures of all of the aforementioned references are herebyincorporated by reference.

The term “interleukin-1 inhibitor” refers to a polypeptide capable ofspecifically preventing activation of cellular receptors to IL-1, whichmay result from any number of mechanisms. Such mechanisms includedownregulating IL-1 production, binding free IL-1, interfering with IL-1binding to its receptor, interfering with formation of the IL-1 receptorcomplex (i.e., association of IL-1 receptor with IL-1 receptor accessoryprotein), or interfering with modulation of IL-1 signaling after bindingto its receptor. Classes of interleukin-1 inhibitors include:

interleukin-1 receptor antagonists such as IL-1ra, as described below;

CDRs or entire variable regions of anti-IL-1 receptor monoclonalantibodies (e.g., EP 623674), the disclosure of which is herebyincorporated by reference;

IL-1 binding proteins such as soluble IL-1 receptors (e.g., U.S. Pat.No. 5,492,888, U.S. Pat. No. 5,488,032, and U.S. Pat. No. 5,464,937,U.S. Pat. No. 5,319,071, and U.S. Pat. No. 5,180,812, the disclosures ofwhich are hereby incorporated by reference);

CDRs or entire variable regions of anti-IL-1 monoclonal antibodies(e.g., WO 9501997, WO 9402627, WO 9006371, U.S. Pat. No. 4,935,343, EP364778, EP 267611 and EP 220063, the disclosures of which are herebyincorporated by reference);

IL-1 receptor accessory proteins and antibodies thereto (e.g., WO96/23067, the disclosure of which is hereby incorporated by reference);

inhibitors of interleukin-1 beta converting enzyme (ICE) or caspase I,which can be used to inhibit IL-1 beta production and secretion;

interleukin-1 beta protease inhibitors;

and other compounds and proteins which block in vivo synthesis orextracellular release of IL-1.

Exemplary IL-1 inhibitors are disclosed in the following references:

U.S. Pat. Nos. 5,747,444; 5,359,032; 5,608,035; 5,843,905; 5,359,032;5,866,576; 5,869,660; 5,869,315; 5,872,095; 5,955,480

International (WO) patent applications 98/21957, 96/09323, 91/17184,96/40907, 98/32733, 98/42325, 98/44940, 98/47892, 98/56377, 99/03837,99/06426, 99/06042, 91/17249, 98/32733, 98/17661, 97/08174, 95/34326,99/36426, and 99/36415.

European (EP) patent applications 534978 and 894795.

French patent application FR 2762514.

The disclosures of all of the aforementioned references are herebyincorporated by reference.

For purposes of the present invention, IL-1ra and variants andderivatives thereof as discussed hereinafter are collectively termed“IL-1ra protein(s)”. The molecules described in the above references andthe variants and derivatives thereof discussed hereinafter arecollectively termed “IL-1 inhibitors.”

The term “TPO-mimetic peptide” comprises peptides that can be identifiedor derived as described in Cwirla et al. (1997), Science 276: 1696-9,U.S. Pat. Nos. 5,869,451 and 5,932,946 and any other reference in Table2 identified as having TPO-mimetic subject matter, as well as the U.S.patent application, “Thrombopoietic Compounds,” filed on Oct. 22, 1999and hereby incorporated by reference. Those of ordinary skill in the artappreciate that each of these references enables one to select differentpeptides than actually disclosed therein by following the disclosedprocedures with different peptide libraries.

The term “leptin protein” refers to native leptin protein and portionsof native leptin protein that retain its anti-diabetes or anti-obesityactivity. Exemplary leptin protein sequence is disclosed in PCT/US97/23183, filed Dec. 11, 1997, which is hereby incorporated byreference.

Additionally, physiologically acceptable salts of the compounds of thisinvention are also encompassed herein. By “physiologically acceptablesalts” is meant any salts that are known or later discovered to bepharmaceutically acceptable. Some specific examples are: acetate;trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide;sulfate; citrate; tartrate; glycolate; and oxalate.

Structure of Compounds

In General. The process of this invention is useful in preparation ofcompositions of matter in which an Fc domain may be attached to thepharmacologically active molecule through the molecule's N-terminus orC-terminus. Thus, the Fc fusion molecules of this invention may bedescribed by the following formula I:(X¹)_(a)—F¹—(X²)_(b)  Iwherein:

F¹ is an Fc domain;

X¹ and X² are each independently selected from -(L¹)_(c)-P¹ and-(L¹)_(c)-P¹-(L²)_(d)-P²;

P¹ and P² are each independently sequences of pharmacologically activepeptides or polypeptides;

L¹ and L² are each independently linkers; and

a, b, c, and d are each independently 0 or 1, provided that at least oneof a and b is 1.

Thus, compound I comprises preferred compounds of the formulaeX¹—F¹  IIand multimers thereof wherein F¹ is attached at the C-terminus of X¹;F¹—X²  IIIand multimers thereof wherein F¹ is attached at the N-terminus of X²;F¹-(L¹)_(c)-P¹  IVand multimers thereof wherein F¹ is attached at the N-terminus of-(L¹)_(c)-P¹;andF¹-(L¹)_(c)-P¹-(L²)_(d)-P²  Vand multimers thereof wherein F¹ is attached at the N-terminus of-L¹-P¹-L²-P².

Peptides. Any number of peptides may be used in conjunction with thepresent invention. Of particular interest are peptides that mimic theactivity of EPO, TPO, growth hormone, G-CSF, GM-CSF, IL-1ra, leptin,CTLA4, TRAIL, TGF-α, and TGF-β. Peptide antagonists are also ofinterest, particularly those antagonistic to the activity of TNF,leptin, any of the interleukins (IL-1, 2, 3, . . . ), and proteinsinvolved in complement activation (e.g., C3b). These peptides may bediscovered by methods described in the references cited in thisspecification and other references.

Phage display, in particular, is useful in generating peptides for usein the present invention. It has been stated that affinity selectionfrom libraries of random peptides can be used to identify peptideligands for any site of any gene product. Dedman et al. (1993), J. Biol.Chem. 268:23025-30. Phage display is particularly well suited foridentifying peptides that bind to such proteins of interest as cellsurface receptors or any proteins having linear epitopes. Wilson et al.(1998), Can. J. Microbiol. 44:313-29; Kay et al. (1998), Drug Disc.Today 3:370-8. Such proteins are extensively reviewed in Herz et al.(1997), J. Receptor& Signal Transduction Res. 17(5):671-776, which ishereby incorporated by reference. Such proteins of interest arepreferred for use in this invention.

Numerous peptides of interest are described in the U.S. patentapplication entitled, “Modified Peptides as Therapeutic Agents,” filedOct. 22, 1999, which is hereby incorporated by reference.

Polypeptides. Numerous polypeptides suitable for use with the subjectinvention are known in the art. Suitable proteins include hormones(e.g., growth hormone), cytokines (e.g., IL-1ra), and soluble receptors(e.g., tumor necrosis factor receptors I and II). Exemplary polypeptidesare those listed in Table 1 (see above).

Fc fusion. An Fc domain may be fused to the N or C termini of thepharmacologically active molecule or at both the N and C termini.

As noted above, Fc variants are suitable vehicles within the scope ofthis invention. A native Fc may be extensively modified to form an Fcvariant in accordance with this invention, provided binding to thesalvage receptor is maintained; see, for example WO 97/34631 and WO96/32478. In such Fc variants, one may remove one or more sites of anative Fc that provide structural features or functional activity notrequired by the fusion molecules of this invention. One may remove thesesites by, for example, substituting or deleting residues, insertingresidues into the site, or truncating portions containing the site. Theinserted or substituted residues may also be altered amino acids, suchas peptidomimetics or D-amino acids. Fc variants may be desirable for anumber of reasons, several of which are described below. Exemplary Fcvariants include molecules and sequences in which:

-   -   1. Sites involved in disulfide bond formation are removed. Such        removal may avoid reaction with other cysteine-containing        proteins present in the host cell used to produce the molecules        of the invention. For this purpose, the cysteine-containing        segment at the N-terminus may be truncated or cysteine residues        may be deleted or substituted with other amino acids (e.g.,        alanyl, seryl). In particular, one may truncate the N-terminal        20-amino acid segment of SEQ ID NO: 2 or delete or substitute        the cysteine residues at positions 7 and 10 of SEQ ID NO: 2.        Even when cysteine residues are removed, the single chain Fc        domains can still form a dimeric Fc domain that is held together        non-covalently.    -   2. A native Fc is modified to make it more compatible with a        selected host cell. For example, one may remove the PA sequence        near the N-terminus of a typical native Fc, which may be        recognized by a digestive enzyme in E. coli such as proline        iminopeptidase. One may also add an N-terminal methionine        residue, especially when the molecule is expressed recombinantly        in a bacterial cell such as E. coli. The Fc domain of SEQ ID NO:        2 (FIG. 5) is one such Fc variant.    -   3. A portion of the N-terminus of a native Fc is removed to        prevent N-terminal heterogeneity when expressed in a selected        host cell. For this purpose, one may delete any of the first 20        amino acid residues at the N-terminus, particularly those at        positions 1, 2, 3, 4 and 5.    -   4. One or more glycosylation sites are removed. Residues that        are typically glycosylated (e.g., asparagine) may confer        cytolytic response. Such residues may be deleted or substituted        with unglycosylated residues (e.g., alanine).    -   5. Sites involved in interaction with complement, such as the        Clq binding site, are removed. For example, one may delete or        substitute the EKK sequence of human IgG1. Complement        recruitment may not be advantageous for the molecules of this        invention and so may be avoided with such an Fc variant.    -   6. Sites are removed that affect binding to Fc receptors other        than a salvage receptor. A native Fc may have sites for        interaction with certain white blood cells that are not required        for the fusion molecules of the present invention and so may be        removed.    -   7. The ADCC site is removed. ADCC sites are known in the art;        see, for example, Molec. Immunol. 29 (5):633-9 (1992) with        regard to ADCC sites in IgG1. These sites, as well, are not        required for the fusion molecules of the present invention and        so may be removed.    -   8. When the native Fc is derived from a non-human antibody, the        native Fc may be humanized. Typically, to humanize a native Fc,        one will substitute selected residues in the non-human native Fc        with residues that are normally found in human native Fc.        Techniques for antibody humanization are well known in the art.

Linkers. Any “linker” group is optional. When present, its chemicalstructure is not critical, since it serves primarily as a spacer. Thelinker is preferably made up of amino acids linked together by peptidebonds. Thus, in preferred embodiments, the linker is made up of from 1to 20 amino acids linked by peptide bonds, wherein the amino acids areselected from the 20 naturally occurring amino acids. Some of theseamino acids may be glycosylated, as is well understood by those in theart. In a more preferred embodiment, the 1 to 20 amino acids areselected from glycine, alanine, proline, asparagine, glutamine, andlysine. Even more preferably, a linker is made up of a majority of aminoacids that are sterically unhindered, such as glycine and alanine. Thus,preferred linkers are polyglycines, poly(Gly-Ala), and polyalanines.Other specific examples of linkers are:

(Gly)₃Lys(Gly)₄; (SEQ ID NO: 3) (Gly)₃AsnGlySer(Gly)₂; (SEQ ID NO: 4)(Gly)₃Cys(Gly)₄; (SEQ ID NO: 5) and GlyProAsnGlyGly. (SEQ ID NO: 6)To explain the above nomenclature, for example, (Gly)₃Lys(Gly)₄ meansGly-Gly-Gly-Lys-Gly-Gly-Gly-Gly. Combinations of Gly and Ala are alsopreferred. The linkers shown here are exemplary; linkers within thescope of this invention may be much longer and may include otherresidues.

Non-peptide linkers are also possible. For example, alkyl linkers suchas —NH—(CH₂)_(s)—C(O)—, wherein s=2-20 could be used. These alkyllinkers may further be substituted by any non-sterically hindering groupsuch as lower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl, Br),CN, NH₂, phenyl, etc. An exemplary non-peptide linker is a PEG linker,

wherein n is such that the linker has a molecular weight of 100 to 5000kD, preferably 100 to 500 kD. The peptide linkers may be altered to formderivatives in the same manner as described above.

Derivatives. The inventors also contemplate derivatizing the peptide,polypeptide and/or Fc portion of the compounds. Such derivatives mayimprove the solubility, absorption, biological half life, and the likeof the compounds. The moieties may alternatively eliminate or attenuateany undesirable side-effect of the compounds and the like.

Exemplary derivatives include compounds in which:

-   -   1. The compound or some portion thereof is cyclic. For example,        the pharmacologically active portion (i.e., peptide or        polypeptide) may be modified to contain two or more Cys residues        (e.g., in the linker), which could cyclize by disulfide bond        formation. For citations to references on preparation of        cyclized derivatives, see Table 2.    -   2. The compound is cross-linked or is rendered capable of        cross-linking between molecules. For example, the        pharmacologically active portion may be modified to contain one        Cys residue and thereby be able to form an intermolecular        disulfide bond with a like molecule. The compound may also be        cross-linked through its C-terminus, as in the molecule shown        below.

-   -   3. One or more peptidyl [—C(O)NR—] linkages (bonds) is replaced        by a non-peptidyl linkage. Exemplary non-peptidyl linkages are        —CH₂-carbamate [—CH₂—OC(O)N—], phosphonate, —CH₂-sulfonamide        [—CH₂—S(O)₂NR—], urea [—NHC(O)NH—], —CH₂-secondary amine, and        alkylated peptide [—C(O)NR⁶— wherein R⁶ is lower alkyl].    -   4. The N-terminus is derivatized. Typically, the N-terminus may        be acylated or modified to a substituted amine. Exemplary        N-terminal derivative groups include —NRR¹ (other than —NH₂),        —NRC(O)R¹,        -   —NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR¹, succinimide, or            benzyloxycarbonyl—NH—(CBZ—NH—), wherein R and R¹ are each            independently hydrogen or lower alkyl and wherein the phenyl            ring may be substituted with 1 to 3 substituents selected            from the group consisting of C₁-C₄ alkyl, C₁-C₄ alkoxy,            chloro, and bromo.    -   5. The free C-terminus is derivatized. Typically, the C-terminus        is esterified or amidated. Exemplary C-terminal derivative        groups include, for example, —C(O)R² wherein R² is lower alkoxy        or —NR³R⁴ wherein R³ and R⁴ are independently hydrogen or C₁-C₈        alkyl (preferably C₁-C₄ alkyl).    -   6. A disulfide bond is replaced with another, preferably more        stable, cross-linking moiety (e.g., an alkylene). See, e.g.,        Bhatnagar et al. (1996), J. Med. Chem. 39:3814-9; Alberts et        al. (1993) Thirteenth Am. Pep. Symp., 357-9.    -   7. One or more individual amino acid residues is modified.        Various derivatizing agents are known to react specifically with        selected sidechains or terminal residues, as described in detail        below.

Lysinyl residues and amino terminal residues may be reacted withsuccinic or other carboxylic acid anhydrides, which reverse the chargeof the lysinyl residues. Other suitable reagents for derivatizingalpha-amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with any one or combinationof several conventional reagents, including phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginyl residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas the arginine epsilon-amino group.

Specific modification of tyrosyl residues has been studied extensively,with particular interest in introducing spectral labels into tyrosylresidues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizole andtetranitromethane are used to form O-acetyl tyrosyl species and 3-nitroderivatives, respectively.

Carboxyl sidechain groups (aspartyl or glutamyl) may be selectivelymodified by reaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Cysteinyl residues can be replaced by amino acid residues or othermoieties either to eliminate disulfide bonding or, conversely, tostabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med.Chem. 39:3814-9.

Derivatization with bifunctional agents is useful for cross-linking thepeptides or their functional derivatives to a water-insoluble supportmatrix or to other macromolecular vehicles. Commonly used cross-linkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Carbohydrate (oligosaccharide) groups may conveniently be attached tosites that are known to be glycosylation sites in proteins. Generally,O-linked oligosaccharides are attached to serine (Ser) or threonine(Thr) residues while N-linked oligosaccharides are attached toasparagine (Asn) residues when they are part of the sequenceAsn-X-Ser/Thr, where X can be any amino acid except proline. X ispreferably one of the 19 naturally occurring amino acids other thanproline. The structures of N-linked and O-linked oligosaccharides andthe sugar residues found in each type are different. One type of sugarthat is commonly found on both is N-acetylneuraminic acid (referred toas sialic acid). Sialic acid is usually the terminal residue of bothN-linked and O-linked linked oligosaccharides and, by virtue of itsnegative charge, may confer acidic properties to the glycosylatedcompound. Such site(s) may be incorporated in the linker of thecompounds of this invention and are preferably glycosylated by a cellduring recombinant production of the polypeptide compounds (e.g., inmammalian cells such as CHO, BHK, COS). However, such sites may furtherbe glycosylated by synthetic or semi-synthetic procedures known in theart.

Other possible modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, oxidation of the sulfur atom in Cys, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains.Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman &Co., San Francisco), pp. 79-86 (1983).

Compounds prepared in the process of the present invention may bechanged at the DNA level, as well. The DNA sequence of any portion ofthe compound may be changed to codons more compatible with the chosenhost cell. For E. coli, which is the preferred host cell, optimizedcodons are known in the art. Codons may be substituted to eliminaterestriction sites or to include silent restriction sites, which may aidin processing of the DNA in the selected host cell. The vehicle, linkerand peptide DNA sequences may be modified to include any of theforegoing sequence changes.

Methods of Making

The compounds prepared in the process of this invention largely may bemade in transformed host cells using recombinant DNA techniques. To doso, a recombinant DNA molecule coding for the peptide is prepared.Methods of preparing such DNA molecules are well known in the art. Forinstance, sequences coding for the peptides could be excised from DNAusing suitable restriction enzymes. Alternatively, the DNA moleculecould be synthesized using chemical synthesis techniques, such as thephosphoramidate method. Also, a combination of these techniques could beused.

This invention also contemplates that a vector capable of expressing themolecules in an appropriate host. The vector comprises the DNA moleculethat codes for the molecule operatively linked to appropriate expressioncontrol sequences. Methods of effecting this operative linking, eitherbefore or after the DNA molecule is inserted into the vector, are wellknown. Expression control sequences include promoters, activators,enhancers, operators, ribosomal binding sites, start signals, stopsignals, cap signals, polyadenylation signals, and other signalsinvolved with the control of transcription or translation.

The resulting vector having the DNA molecule thereon is used totransform an appropriate host. This transformation may be performedusing methods well known in the art.

Any of a large number of available and well-known host cells may be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityof the peptides encoded by the DNA molecule, rate of transformation,ease of recovery of the peptides, expression characteristics, bio-safetyand costs. A balance of these factors must be struck with theunderstanding that not all hosts may be equally effective for theexpression of a particular DNA sequence. Within these generalguidelines, useful microbial hosts include bacteria (such as E. colisp.), yeast (such as Saccharomyces sp.) and other fungi, insects,plants, mammalian (including human) cells in culture, or other hostsknown in the art.

Next, the transformed host is cultured and purified. Host cells may becultured under conventional fermentation conditions so that the desiredcompounds are expressed. Such fermentation conditions are well known inthe art. Finally, the peptides are purified from culture by methods wellknown in the art.

The compounds may also be made by synthetic methods. For example, solidphase synthesis techniques may be used. Suitable techniques are wellknown in the art, and include those described in Merrifield (1973),Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.);Merrifield (1963), J. Am. Chem. Soc. 85:2149; Davis et al. (1985),Biochem. Intl. 10:394-414; Stewart and Young (1969), Solid Phase PeptideSynthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins(3rd ed.)2:105-253; and Erickson et al. (1976), The Proteins (3rd ed.)2:257-527. Solid phase synthesis is the preferred technique of makingindividual peptides since it is the most cost-effective method of makingsmall peptides.

Compounds that contain derivatized peptides or which contain non-peptidegroups may be synthesized by well-known organic chemistry techniques.

Uses of the Compounds

In general. The compounds of this invention have pharmacologic activityresulting from the pharmacologically active molecules to which the Fc isattached. The activity of these compounds can be measured by assaysknown in the art.

In addition to therapeutic uses, the compounds of the present inventionmay also be useful in diagnosing diseases characterized by dysfunctionof an associated protein of interest. In one embodiment, a method ofdetecting in a biological sample a protein of interest (e.g., areceptor) that is capable of being activated comprising the steps of:(a) contacting the sample with a compound of this invention; and (b)detecting activation of the protein of interest by the compound. Thebiological samples include tissue specimens, intact cells, or extractsthereof. The compounds of this invention may be used as part of adiagnostic kit to detect the presence of their associated proteins ofinterest in a biological sample. Such kits employ the compounds of theinvention having an attached label to allow for detection. The compoundsare useful for identifying normal or abnormal proteins of interest. Forthe EPO-mimetic compounds, for example, presence of abnormal protein ofinterest in a biological sample may be indicative of such disorders asDiamond Blackfan anemia, where it is believed that the EPO receptor isdysfunctional.

Pharmaceutical Compositions

In General. The present invention also provides methods of usingpharmaceutical compositions of the inventive compounds. Suchpharmaceutical compositions may be for administration for injection, orfor oral, pulmonary, nasal, transdermal or other forms ofadministration. In general, the invention encompasses pharmaceuticalcompositions comprising effective amounts of a compound of the inventiontogether with pharmaceutically acceptable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers. Such compositionsinclude diluents of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength; additives such as detergents andsolubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,mannitol); incorporation of the material into particulate preparationsof polymeric compounds such as polylactic acid, polyglycolic acid, etc.or into liposomes. Hyaluronic acid may also be used, and this may havethe effect of promoting sustained duration in the circulation. Suchcompositions may influence the physical state, stability, rate of invivo release, and rate of in vivo clearance of the present proteins andderivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed.(1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which areherein incorporated by reference. The compositions may be prepared inliquid form, or may be in dried powder, such as lyophilized form.Implantable sustained release formulations are also contemplated, as aretransdermal formulations.

Pulmonary delivery forms. Also contemplated herein is pulmonary deliveryof the Fc fusion molecules (or derivatives thereof). The protein (orderivative) is delivered to the lungs of a mammal while inhaling andtraverses across the lung epithelial lining to the blood stream. (Otherreports of this include Adjei et al., Pharma. Res. (1990) 7:565-9; Adjeiet al. (1990), Internatl. J. Pharmaceutics 63:135-44 (leuprolideacetate); Braquet et al. (1989), J. Cardiovasc. Pharmacol. 13(suppl.5):s.143-146 (endothelin-1); Hubbard et al. (1989), Annals Int.Med. 3:206-12 (α1-antitrypsin); Smith et al. (1989), J. Clin. Invest.84:1145-6 (α1-proteinase); Oswein et al. (March 1990), “Aerosolizationof Proteins”, Proc. Symp. Resp. Drug Delivery II, Keystone, Colo.(recombinant human growth hormone); Debs et al. (1988), J. Immunol.140:3482-8 (interferon-γ and tumor necrosis factor α) and Platz et al.,U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor).

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. Some specific examples of commercially availabledevices suitable for the practice of this invention are the Ultraventnebulizer, manufactured by. Mallinckrodt, Inc., St. Louis, Mo.; theAcorn II nebulizer, manufactured by Marquest Medical Products,Englewood, Colo.; the Ventolin metered dose inhaler, manufactured byGlaxo Inc., Research Triangle Park, N.C.; and the Spinhaler powderinhaler, manufactured by Fisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of the inventive compound. Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to diluents, adjuvantsand/or carriers useful in therapy.

The inventive compound should most advantageously be prepared inparticulate form with an average particle size of less than 10 μm (ormicrons), most preferably 0.5 to 5 μm, for most effective delivery tothe distal lung.

Pharmaceutically acceptable carriers include carbohydrates such astrehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Otheringredients for use in formulations may include DPPC, DOPE, DSPC andDOPC. Natural or synthetic surfactants may be used. PEG may be used(even apart from its use in derivatizing the protein or analog).Dextrans, such as cyclodextran, may be used. Bile salts and otherrelated enhancers may be used. Cellulose and cellulose derivatives maybe used. Amino acids may be used, such as use in a buffer formulation.

Also, the use of liposomes, microcapsules or microspheres, inclusioncomplexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the inventive compound dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per mL of solution. The formulation may also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation may also contain asurfactant, to reduce or prevent surface induced aggregation of theprotein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the inventive compoundsuspended in a propellant with the aid of a surfactant. The propellantmay be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing the inventive compound and may alsoinclude a bulking agent, such as lactose, sorbitol, sucrose, mannitol,trehalose, or xylitol in amounts which facilitate dispersal of thepowder from the device, e.g., 50 to 90% by weight of the formulation.

Nasal delivery forms. Nasal delivery of the Fc fusion molecule is alsocontemplated. Nasal delivery allows the passage of the protein to theblood stream directly after administering the therapeutic product to thenose, without the necessity for deposition of the product in the lung.Formulations for nasal delivery include those with dextran orcyclodextran. Delivery via transport across other mucous membranes isalso contemplated.

Dosages. The dosage regimen involved in a method for treating theabove-described conditions will be determined by the attendingphysician, considering various factors which modify the action of drugs,e.g. the age, condition, body weight, sex and diet of the patient, theseverity of any infection, time of administration and other clinicalfactors. Generally, the daily regimen should be in the range of 0.1-1000micrograms of the inventive compound per kilogram of body weight,preferably 0.1-150 micrograms per kilogram.

SPECIFIC PREFERRED EMBODIMENTS Working Examples

The following disclosure(s) are illustrative rather than limiting.

Example 1

Identification of cysteines in a Fc construct of osteoprotegerin by CDAPlabeling and alkaline-induced cleavage with LC-MS analysis

Abstract

Purpose. To assay for cysteines in a Fc construct of Osteoprotegerinstored in acidic media and to ascertain their location.

Methods. The reagent 1-cyano-4-dimethylamino-pyridiniumtetrafluoroborate (CDAP) was employed to selectively cyanylate stablefree-sulfhydryl groups at pH 4. Cyanylation reactions were analyzed byHPLC coupled with mass-spectral analysis (LC-MS). Reversed-phase HPLCwas used to separate component peaks from the native and CDAP-reactedsamples. The reversed-phase samples were collected, introduced into asolution of aqueous ammonium hydroxide plus guanidine hydrochloride andsubsequently analyzed by LC-MS. Results. Reversed-phase HPLC analysis ofFc-Osteoprotegerin gave two peaks. LC-MS of the primary CDAP reactionrevealed selective cyanylation of two-sulfhydryl groups in a peak thatrepresents approximately 10% of the proteinaceous material; the peakrepresenting approximately 90% of the material was not cyanylated afterreaction with CDAP. Analysis of the ammonium/guanidine treated isoformsby LC-MS detailed the location of two free cysteine-sulfhydryl groups.

Conclusions. A reversed-phase separable form of Fc-Osteoprotegerincontains two free sulfhydryl groups. CDAP is an effective reagent forcharacterizing the free-sulfhydryl groups of proteins in acidicconditions.

Introduction

Fc-OPG, a construct of the naturally occurring cytokine Osteoprotegerin(OPG), is under clinical investigation for utility in the treatment ofosteoporosis. Fc-OPG is a homodimeric, high molecular weight protein (91kD) containing 24 cysteines in each monomer sequence. All cysteines inthe dimer are covalently associated through 24 disulfide bonds (orcystine). These covalent linkages play a crucial role in maintaining thetertiary and quaternary structure of the protein. Therefore, unpairedcysteines may result in a decrease in biological activity, haveimmunogenic effects, or be a precursor to further physical and chemicalinstability (oxidation and aggregation).

Monitoring the state of cysteine side-chains—in the disulfide form ofcystine or sulfhydryl of cysteine—is commonly accomplished by labelingthe free sulfhydryl groups with a thiol specific fluorophor or molecularweight tag under alkaline conditions. Given the labile nature ofcysteine-sulfhydryl groups and cystine-disulfides in alkaline aqueousmedia (pKa's˜8), quantitative detection of free cysteine-sulfhydryl issubject to inaccuracy resulting from cystine reduction at high pH andcysteine related disulfide formation. Alkaline condition labelingmethods may not be suitable for estimating the free-cysteine-sulfhydrylpresent in acidic formulations of proteins.

A reverse-phase HPLC separable isoform of Fc-OPG was observed to bereactive to the addition of aqueous CuCl₂. After reaction with CuCl₂,the isoform eluted with the same retention time as the main peak in thereverse-phase chromatogram. The nature of this reactivity led tospeculation that the isoform was representative of free-sulfhydrylgroups in the protein that were converted to cystine with the aid of acopper oxidant. Numerous techniques were employed to compare the numberof free cysteine-sulfhydryl groups present in the main peak versus theisoform. No differences were found. A commonality between all of thetechniques was the use of neutral to alkaline reaction conditions.

This example presents the characterization of the isoform using thereagent 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP). Thedetection and identification of unpaired cysteine residues present inacidic media is made possible by the cyanylation of free cysteinesulfhydryl groups by CDAP (FIG. 6) and subsequent peptide bond cleavageat the cyanylated cysteine residues (FIG. 7).

Materials

The Fc construct of OPG can be prepared by processes known in the art.CDAP can be purchased from Sigma-Aldrich (St. Louis, Mo).

Methods

RP-HPLC: Samples were injected into a Zorbax 300SB C8 4.6 mm×25 cmcolumn and eluted with a gradient of 0.1% TFA and 0.1% TFA in 90% ACN at60° C. on a Hewlett-Packard 1100 or 1090 at 215 nm.

Electrospray Mass Spectrometry: All mass spectrometry was performed on aPerkin-Elmer/Sciex AP100 spectrometer using conditions consistent withionization of peptides and proteins.

Copper Treatment: Ten mg/ml protein in 10 mM sodium acetate and 5%sorbitol was treated with 50 mM CuCl₂ (final pH ˜4).

Cyanylation Conditions: A solution of 50 mg/mL of CDAP in 0.5 M HCl wasprepared. 50 μL of the CDAP solution was added to 500 μL of 10 mg/mL ofprotein in pH 5 acetate (10 mM) and 5% sorbitol. 5 μL of 0.1 N NaOH wasadded to the protein-CDAP mixture and vortexed.

Cleavage Conditions: The cyanylated protein was separated from nativematerial by reverse-phase HPLC and both peaks were collected. Thefractions were reduced in volume by approximately two-thirds undervacuum then treated with a solution of 1.5 M NH₄OH, 4 M GdHCl and heatedat 37° C. for 90 minutes.

Discussion

Cyanylation of two cysteine sulfhydryl groups in Fc-OPG would give amass increase of 50 daltons resulting from the addition of two 26 daltoncyanide groups and the assumed loss of two protons from the cysteineside chains. The selective cyanylation of the RP-HPLC isoform is evidentin the ˜50 dalton mass increase detected for that peak; the main peakmass was unmodified. The nature of the isoform was further indicated bythe observation that the isoform was no-longer responsive to theaddition of CuCl₂. A pair of cyanylated cysteines would not be competentfor disulfide formation via CuCl₂ oxidation (FIG. 8).

The cleavage pattern observed for the CDAP reacted and ammoniumhydroxide treated isoform differed greatly from that of the main peak.The isoform cleavage reaction produced three distinct peaks and the mainpeak reaction resulted in one peak (FIG. 9). Mass spectrometry of theisoform cleavage products established their identity (Table 1). The peakwith mass of 22,270 amu correlated with a fragment of the moleculeconsisting of the entire amino acid sequence C-terminal of the lastcysteine in the Fc sequence (FIG. 9, peak 2). The mass found for peak 3in FIG. 4, 6616.8 amu, is equivalent to the mass of a Fc-domaindisulfide loop corresponding to the Fc sequence found N-terminally tothe fragment determined for peak 2. Peak 4 (FIG. 9) correlates with theremaining piece of the Fc-OPG construct. These results reveal twofree-cysteine-sulfhydryl groups in the Fc domain of one of the twomonomers (FIG. 10).

In subsequent experiments, the RP-HPLC isoform was converted to mainpeak under alkaline-denaturing conditions (data not shown). Apparently,formation of cystine in the isoform was rapid enough in those alkalineconditions to go undetected by other techniques (Ellman's reaction andpeptide mapping). The site specificity and acid-condition-compatibilityof CDAP for labeling cysteine residues made possible thecharacterization of this protein isoform.

Conclusions

A reversed-phase HPLC separable isoform of Fc-OPG contains two freesulfhydryl groups. The detection of free cysteine sulfhydryl groups in aprotein can be complicated by their transient nature in the alkalineconditions typically used for analysis. CDAP is an effective reagent forcharacterizing the free-sulfhydryl groups of proteins in acidicconditions.

Example 2

Purpose. Significant differences were observed in the stability ofFc-OPG as a function of copper treatment of the bulk protein, whereFc-OPG treated with copper was significantly more stable than Fc-OPGthat was not. Specifically, the Fc-OPG that was not treated with copperwas more prone to aggregation than the copper-treated Fc-OPG. Theimproved stability of Fc-OPG is thought to be due to the conversion ofan unstable fraction of Fc-OPG with incomplete disulfide structure to aform with intact disulfide bonding upon treatment with copper ion.

Methods. Reversed-phase chromatography: Reversed-phase chromatographywas performed on a Hewlett-Packard 1100 or 1090 HPLC system equippedwith a diode-array detector and controlled by Chemstation Software.Fc-OPG samples were analyzed on a Zorbax (4.6 mm×25 cm) 300 SB column.The column is initially equilibrated at 90% buffer A (HPLC grade watercontaining 0.1% TFA). The samples were eluted with a linear gradient of4.2% buffer B/min (90% acetonitrile, 10% HPLC grade water and 0.1% TFA)for 6 minutes then 0.11% buffer B/min over 37 minutes. The column washeated at 60° C. and elution was monitored 215 nm at a flow rate of 0.5ml/min. Both copper and non-copper treated Fc-OPG (10 mg/mL) wereformulated in 10 mM sodium acetate, pH 5.0, containing 5% sorbitol.Samples were incubated at 29° C. and analyzed over time.

Conclusions. As has been previously shown, CuCl₂ catalyzes the formationof cystine from two unpaired cysteines in Fc-OPG. The reversed-phaseprofiles of a Fc-OPG preparation after copper treatment versus one whichwas untreated are compared in FIG. 11. The prominent post-peak in thechromatogram of the untreated sample is due to the population of Fc-OPGmolecules with unpaired cysteines. The stability of these Fc-OPGpreparations was examined as a function of copper treatment bysize-exclusion HPLC. The chromatograms of untreated and copper-treatedFc-OPG incubated at 29° C. for 2 years are shown in FIG. 12; an increasein high molecular weight aggregate and dimer peaks are evident in theuntreated sample.

ABBREVIATIONS

The abbreviations used throughout this specification are defined asfollows, unless otherwise defined in specific instances:

-   -   CDAP 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate    -   HPLC high performance liquid chromatography    -   ITZ iminothiazolidine    -   OPG osteoprotegerin    -   RP reversed-phase    -   TNF tumor necrosis factor    -   TPO thrombopoeitin

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto, without departing from the spirit and scope of theinvention as set forth herein.

1. A process for preparing a pharmacologically active compound, whichcomprises: (a) preparing an active compound which is a fusion moleculecomprising a pharmacologically active domain and an Fc domain; (b)incubating the active compound with copper (II) chloride (CuCl₂) at aconcentration from about 10 mM to 50 mM; and (c) subsequently isolatingthe active compound.
 2. The process of claim 1, wherein thepharmacologically active compound is prepared in Escherichia coli. 3.The process of claim 2, wherein the CuCl₂ is used in a concentration ofabout 10 mM.
 4. The process of claim 1, wherein the pharmacologicallyactive compound is prepared in CHO cells.
 5. The process of claim 4,wherein the CuCl₂ is used in a concentration of about 30 mM.
 6. Theprocess of claim 1, wherein the pharmacologically active domaincomprises the sequence of an OPG protein.
 7. The process of claim 1,wherein the pharmacologically active domain comprises the sequence of aleptin protein.
 8. The process of claim 1, wherein the pharmacologicallyactive domain comprises the sequence of a soluble TNF receptor.
 9. Theprocess of claim 1, wherein the pharmacologically active domaincomprises the sequence of a soluble IL-1 receptor.
 10. The process ofclaim 1, wherein the pharmacologically active domain comprises thesequence of an IL-1ra protein.
 11. The process of claim 1, wherein theFc domain is an IgG1 Fc domain.
 12. The process of claim 1, wherein theFc domain comprises the sequence of SEQ ID NO:2.